Bottom Line:
We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA.We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP.Three of them also protect from AHA.

Background: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast.

Results: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism.

Conclusion: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

Mentions:
Based on our data, we propose that, in yeast, the major base analogs protective mechanism is a control of the quality of DNA precursor pool that prevents incorporation of base analogs into DNA. This mechanism may work at several levels: transport of analogs into cells, detoxification of analogs by metabolic enzymes, maintenance of nucleotide pools, and fidelity control of DNA replication (Fig. 4). HAP and AHA are likely transported into the yeast cell by the same permeases, which are involved in transport of natural purines. One candidate is purine-cytosine permease, Fcy2p, a major purine (adenine, guanine, and hypoxanthine) and cytosine transporter in yeast [31]. According to our unpublished data, fcy2 mutants are resistant to HAP. Thus, the active transport of HAP is the first critical step in the HAP mutagenic pathway. The next step is a conversion of the base analog to the corresponding ribonucleoside monophosphate by enzymes of the purine salvage pathway. Previously we observed that the inactivation of the APT1 gene, encoding adenine phosphoribosyl transferase, led to a severe decrease of the mutagenic effect of HAP [16], suggesting that this enzyme plays a key role in the biosynthesis of HAP-riboside-5'-monophosphate (HAPMP). HAPMP then may be converted to the corresponding nucleoside triphosphate, which could be ambiguously incorporated into DNA by DNA polymerases and provoke replication errors in the subsequent replication cycles [13].

Mentions:
Based on our data, we propose that, in yeast, the major base analogs protective mechanism is a control of the quality of DNA precursor pool that prevents incorporation of base analogs into DNA. This mechanism may work at several levels: transport of analogs into cells, detoxification of analogs by metabolic enzymes, maintenance of nucleotide pools, and fidelity control of DNA replication (Fig. 4). HAP and AHA are likely transported into the yeast cell by the same permeases, which are involved in transport of natural purines. One candidate is purine-cytosine permease, Fcy2p, a major purine (adenine, guanine, and hypoxanthine) and cytosine transporter in yeast [31]. According to our unpublished data, fcy2 mutants are resistant to HAP. Thus, the active transport of HAP is the first critical step in the HAP mutagenic pathway. The next step is a conversion of the base analog to the corresponding ribonucleoside monophosphate by enzymes of the purine salvage pathway. Previously we observed that the inactivation of the APT1 gene, encoding adenine phosphoribosyl transferase, led to a severe decrease of the mutagenic effect of HAP [16], suggesting that this enzyme plays a key role in the biosynthesis of HAP-riboside-5'-monophosphate (HAPMP). HAPMP then may be converted to the corresponding nucleoside triphosphate, which could be ambiguously incorporated into DNA by DNA polymerases and provoke replication errors in the subsequent replication cycles [13].

Bottom Line:
We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA.We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP.Three of them also protect from AHA.

Background: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast.

Results: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism.

Conclusion: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.